We recently discovered the tetracycline destructases, a new family of flavoenzymes capable of degrading tetracycline through novel mechanisms, distinct from the canonical resistance strategies of efflux or ribosomal protection. The rationale for the proposed research is that increasing antibiotic resistance in pathogens is a public health emergency, fed by a potent reservoir of under-characterized antibiotic resistance genes found in benign environmental bacteria. The central motivation for this proposal is to comprehensively understand and model the sequence-structure-function-substrate space for this novel class of tetracycline resistance enzymes, so as to design strategies to mitigate their clinical impact before they widely disseminate into important pathogens. Guided by strong preliminary data, this proposal will pursue three independent yet complementary specific aims: 1) Elucidate the mechanism of action of the tetracycline destructases, 2) Characterize the evolutionary landscape of the tetracycline destructases, and 3) Rationally design and synthesize tetracycline destructase inhibitors. The first aim will test the hypothesis that the tetracycline destructases degrade tetracycline via two independent and potentially novel mechanisms that are controlled at the sequence- structure level. Crystallography and enzymology will be used to comprehensively elucidate the structure and mechanism of action of the tetracycline destructases. The second aim examines the hypothesis that the tetracycline destructases originated as biosynthetic flavoenzymes, and are poised to become clinical threats. This aim will explore the sequence-structure-function space, model the fitness landscape, and elucidate cross-resistance mechanism interactions of the tetracycline destructases. The third aim proposes the hypothesis that susceptibility to tetracycline can be restored by inhibition of the tetracycline destructases. This will be achieved by the structure-guided design, synthesis, and characterization of tetracycline destructase inhibitors. This proposal is innovative because our integrated and complementary research team will test novel concepts in emerging antibiotic resistance, and apply multi-disciplinary technological innovation to comprehensively study and target tetracycline destructases. The proposed research is significant because in the United States alone, more than two million people every year suffer an antibiotic-resistant infection, leading to at least 23,000 deaths and an estimated $55 billion of excess healthcare-related costs. Antibiotic resistance has steadily increased in pathogenic and benign bacteria since the introduction of antibiotics to the clinic and in agriculture. Tetracycline destructases are likely candidates for dissemination to the clinic, potentially compromising new tetracycline derivatives and motivating deep mechanistic analysis of their activities. The proposed research is impactful because it will (1) advance fundamental understanding of the evolutionary origins of anti- biotic resistance, (2) assess the clinical threat posed by environmental resistance genes, and (3) devise inhibitor-based solutions to the problem of antibiotic resistance through the lens of the tetracycline destructases.